Stable pharmaceutical compsition containing factor VIII

ABSTRACT

The invention relates to a stable solid pharmaceutical composition comprising factor VIII. Such a composition is devoid of amino acids and comprises: (a) factor VIII; (b) a surfactant; (c) calcium chloride; (d) sucrose; (e) sodium chloride; (f) trisodium citrate; and (g) a buffer devoid of amino acids; and has a pH from 6 to 8 prior to lyophilisation and after reconstitution in water for injection. The invention also relates to the liquid pharmaceutical composition obtainable after dilution of said stable solid pharmaceutical composition with sterile water optionally containing sodium chloride.

The invention relates to a new stable pharmaceutical compositioncontaining factor VIII.

Factor VIII is a well-known plasma protein that is essential to theblood clotting process and is therefore used in the treatment ofhaemophilia

Several forms of factor VIII have been used or are intended to be usedas active principles for treating haemophilia These include human factorVIII (like the active principles of Humate® P, Monoclate® P, lmmunate®or Hemofil® M), recombinant human factor VIII (like r-VIII SQ which isdescribed in PCT patent application WO 91/09122 (the active principle ofReFacto®) or the active principles of Kogenate® or Recombinate®),porcine factor VIII (which is the active principle of the productHyate:C® sold by Ipsen, Inc., USA) or recombinant porcine factor VIII(e.g. a modified B-domainless form of porcine factor VIII like the onedisclosed in patent application WO 01/68109 and identified as “POL1212”or the protein of SEQ. ID. NO. 38 of the same patent application).

Formulation stability has always been a problem for the pharmaceuticalindustry dealing with factor VIII pharmaceutical compositions.

Albumin has often been used to stabilise these formulations. However,despite its interesting stabilising effect, albumin presents thedrawback of being expensive and also the risk to carry infectiousspecies like prions. For these reasons, the pharmaceutical industry hasbeen seeking in the recent years to replace albumin by other stabilisingagents in factor VIII pharmaceutical compositions.

Several stable albumin-free pharmaceutical compositions are alreadyknown to the skilled artisan For example:

-   -   U.S. Pat. No. 5,565,427 teaches a stabilised albumin-free        solution with factor VIII:C activity containing factor VIII:C,        an amino acid or one of its salts or homologues and a detergent        (like polysorbate 80 or Tween® 80) or an organic polymer (like        polyethyleneglycol).    -   U.S. Pat. No. 5,605,884 relates to a stable factor VIII        composition comprising factor VIII and a high ionic strength        media, which is preferably consisting of an aqueous solution        comprising a mixture of sodium chloride, calcium chloride and        histidine as buffer ion.    -   U.S. Pat. Nos. 5,763,401 and 5,874,408 both disclose a stable        albumin-free recombinant factor VIII composition comprising        recombinant factor VIII, glycine, histidine, sucrose, sodium        chloride and calcium chloride.    -   U.S. Pat. No. 5,962,650 teaches a stable albumin-free        recombinant factor VIII composition which consists of an aqueous        solution with a reduced concentration of oxygen comprising        recombinant factor VIII, a calcium salt like calcium chloride        and preferably an antioxidant, a non-onic surfactant, sodium or        potassium chloride, an amino acid and a mono- or disaccharide.    -   U.S. Pat. No. 5,972,885 relates to a pharmaceutical formulation        for subcutaneous, intramuscular or intradermal administration        which comprises highly concentrated (at least 1,000 IU/ml)        recombinant factor VIII and, preferably, one or more elements        selected from the group constituted (notably) by sodium or        potassium chloride, calcium chloride, a non-ionic surfactant        (e.g. a poloxamer), a mono- or disaccharide (preferably sucrose)        and antioxidants (e.g. citric acid).    -   PCT patent application WO 89/09784 discloses a method for the        production of heat-stable factor VIII concentrate which        comprises gel filtration of a buffer solution containing said        factor VIII and tris(hydroxymethyl)methylamine, trisodium        citrate, sodium chloride, sucrose and calcium chloride followed        by freeze-drying of the concentrate obtained. The factor VIII        thus produced is able to withstand temperatures of up to 80° C.        for up to 72 hours.    -   PCT patent application WO 94/07510 describes a factor VIII        composition which is stabilised by a non-ionic surfactant (e.g.        a poloxamer like polysorbate 80). Such a composition can also        comprise one or more elements selected from the group        constituted (notably) by sodium or potassium chloride, calcium        chloride, an amino acid, a mono- or disaccharide such as        sucrose,

The Applicant has now unexpectedly discovered that a solidpharmaceutical composition obtainable by lyophilisation of a solutiondevoid of amino acids comprising the following components:

-   (a) factor VIII;-   (b) a surfactant;-   (c) calcium chloride;-   (d) sucrose;-   (e) sodium chloride;-   (f) trisodium citrate; and-   (g) a buffer devoid of amino acids;    said pharmaceutical composition having a pH from 6 to 8 prior to    lyophilisation and after reconstitution in water for injection, also    shows stability over time.

By factor VIII is meant in the present application human factor VIII,recombinant human factor VIM, porcine factor VIM, recombinant porcinefactor VIII or more generally any other recombinant factor VIII that canbe used to replace them.

Preferably, the factor VIII comprised in compositions according to theinvention, will be chosen from porcine factor VIII or recombinantporcine factor VIII. Still more preferably, the factor VIII comprised incompositions according to the invention, will be recombinant porcinefactor VIII, especially a modified B-domainless form of porcine factorVIII like the one disclosed in patent application WO 01/68109, i.e. themodified porcine factor VIII having the amino acid sequence SEQ. ID. NO.1 hereafter: SEQ ID. NO.1: Met Gln Leu Glu Leu Ser Thr Cys Val Phe LeuCys Leu Leu Pro Leu  1               5                  10                  15 Gly Phe SerAla Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser             20                  25                  30 Trp Asp Tyr ArgGln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr         35                  40                  45 Arg Phe Pro Ala ThrAla Pro Gly Ala Leu Pro Leu Gly Pro Ser Val     50                  55                  60 Leu Tyr Lys Lys Thr ValPhe Val Glu Phe Thr Asp Gln Leu Phe Ser 65                  70                  75                  80 Val AlaArg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile                 85                  90                  95 Gln Ala GluVal Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala            100                 105                 110 Ser His Pro ValSer Leu His Ala Val Gly Val Ser Phe Trp Lys Ser        115                 120                 125 Ser Glu Gly Ala GluTyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu    130                 135                 140 Asp Asp Lys Val Leu ProGly Lys Ser Gln Thr Tyr Val Trp Gln Val145                 150                 155                 160 Leu LysGlu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr                165                 170                 175 Ser Tyr LeuSer His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu            180                 185                 190 Ile Gly Ala LeuLeu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg        195                 200                 205 Thr Gln Asn Leu HisGlu Phe Val Leu Leu Phe Ala Val Phe Asp Glu    210                 215                 220 Gly Lys Ser Trp His SerAla Arg Asn Asp Ser Trp Thr Arg Ala Met225                 230                 235                 240 Asp ProAla Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly                245                 250                 255 Tyr Val AsnArg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser            260                 265                 270 Val Tyr Trp HisVal Ile Gly Met Gly Thr Ser Pro Glu Val His Ser        275                 280                 285 Ile Phe Leu Glu GlyHis Thr Phe Leu Val Arg His His Arg Gln Ala    290                 295                 300 Ser Leu Glu Ile Ser ProLeu Thr Phe Leu Thr Ala Gln Thr Phe Leu305                 310                 315                 320 Met AspLeu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His                325                 330                 335 His Gly GlyMet Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu            340                 345                 350 Pro Gln Leu ArgArg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn        355                 360                 365 Leu Tyr Asp Ser AspMet Asp Val Val Arg Leu Asp Gly Asp Asp Val    370                 375                 380 Ser Pro Phe Ile Gln IleArg Ser Val Ala Lys Lys His Pro Lys Thr385                 390                 395                 400 Trp ValHis Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro                405                 410                 415 Ala Val ProSer Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn            420                 425                 430 Ser Gly Pro GlnArg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val        435                 440                 445 Ala Tyr Thr Asp ValThr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu    450                 455                 460 Ser Gly Ile Leu Gly ProLeu Leu Tyr Gly Glu Val Gly Asp Thr Leu465                 470                 475                 480 Leu IleIle Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro                485                 490                 495 His Gly IleThr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys            500                 505                 510 Gly Trp Lys HisLeu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe        515                 520                 525 Lys Tyr Lys Trp ThrVal Thr Val Glu Asp Gly Pro Thr Lys Ser Asp    530                 535                 540 Pro Arg Cys Leu Thr ArgTyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys545                 550                 555                 560 Asp LeuAla Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu                565                 570                 575 Ser Val AspGln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val            580                 585                 590 Ile Leu Phe SerVal Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu        595                 600                 605 Asn Ile Gln Arg PheLeu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp    610                 615                 620 Pro Glu Phe Gln Ala SerAsn Ile Met His Ser Ile Asn Gly Tyr Val625                 630                 635                 640 Phe AspSer Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp                645                 650                 655 Tyr Ile LeuSer Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe            660                 665                 670 Ser Gly Tyr ThrPhe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr        675                 680                 685 Leu Phe Pro Phe SerGly Glu Thr Val Phe Met Ser Met Glu Asn Pro    690                 695                 700 Gly Leu Trp Val Leu GlyCys His Asn Ser Asp Leu Arg Asn Arg Gly705                 710                 715                 720 Met ThrAla Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp                725                 730                 735 Tyr Tyr AspAsn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly            740                 745                 750 Lys Asn Val IleGlu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro        755                 760                 765 Ser Ala Ser Ala ProLys Pro Pro Val Leu Arg Arg His Gln Arg Asp    770                 775                 780 Ile Ser Leu Pro Thr PheGln Pro Glu Glu Asp Lys Met Asp Tyr Asp785                 790                 795                 800 Asp IlePhe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly                805                 810                 815 Glu Asp GluAsn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His            820                 825                 830 Tyr Phe Ile AlaAla Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu        835                 840                 845 Ser Pro Arg Ala LeuArg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg    850                 855                 860 Phe Lys Lys Val Val PheArg Glu Phe Ala Asp Gly Ser Phe Thr Gln865                 870                 875                 880 Pro SerTyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro                885                 890                 895 Tyr Ile ArgAla Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn            900                 905                 910 Gln Ala Ser ArgPro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro        915                 920                 925 Asp Asp Gln Glu GlnGly Ala Glu Pro Arg His Asn Phe Val Gln Pro    930                 935                 940 Asn Glu Thr Arg Thr TyrPhe Trp Lys Val Gln His His Met Ala Pro945                 950                 955                 960 Thr GluAsp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val                965                 970                 975 Asp Leu GluLys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile            980                 985                 990 Cys Arg Ala AsnThr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val        995                1000                1005 Gln Glu Phe Ala LeuPhe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp   1010                1015                1020 Tyr Phe Thr Glu Asn ValGlu Arg Asn Cys Arg Ala Pro Cys His Leu1025               1030                1035                1040 Gln MetGlu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile               1045                1050                1055 Asn Gly TyrVal Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn           1060                1065                1070 Gln Arg Ile ArgTrp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile       1075                1080                1085 His Ser Ile His PheSer Gly His Val Phe Ser Val Arg Lys Lys Glu   1090                1095                1100 Glu Tyr Lys Met Ala ValTyr Asn Leu Tyr Pro Gly Val Phe Glu Thr1105               1110                1115                1120 Val GluMet Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu               1125                1130                1135 Ile Gly GluHis Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr           1140                1145                1150 Ser Lys Glu CysGln Ala Pro Leu Gly Met Ala Ser Gly Arg Ile Arg       1155                1160                1165 Asp Phe Gln Ile ThrAla Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys   1170                1175                1180 Leu Ala Arg Leu His TyrSer Gly Ser Ile Asn Ala Trp Ser Thr Lys1185                1190                1195                1200 Asp ProHis Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile               1205                1210                1215 His Gly IleMet Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr           1220                1225                1230 Ile Ser Gln PheIle Ile Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln       1235                1240                1245 Ser Tyr Arg Gly AsnSer Thr Gly Thr Leu Met Val Phe Phe Gly Asn   1250                1255                1260 Val Asp Ala Ser Gly IleLys His Asn Ile Phe Asn Pro Pro Ile Val1265                1270                1275                1280 Ala ArgTyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr               1285                1290                1295 Leu Arg MetGlu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro           1300                1305                1310 Leu Gly Met GlnAsn Lys Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser       1315                1320                1325 Ser His Leu Ser AsnIle Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg   1330                1335                1340 Leu His Leu Gln Gly ArgThr Asn Ala Trp Arg Pro Arg Val Ser Ser1345                1350                1355                1360 Ala GluGlu Trp Leu Gln Val Asp Leu Gln Lys Thr Val Lys Val Thr               1365                1370                1375 Gly Ile ThrThr Gln Gly Val Lys Ser Leu Leu Ser Ser Met Tyr Val           1380                1385                1390 Lys Glu Phe LeuVal Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu       1395                1400                1405 Phe Leu Gln Asp GlyHis Thr Lys Val Phe Gln Gly Asn Gln Asp Ser   1410                1415                1420 Ser Thr Pro Val Val AsnAla Leu Asp Pro Pro Leu Phe Thr Arg Tyr1425                1430                1435                1440 Leu ArgIle His Pro Thr Ser Trp Ala Gln His Ile Ala Leu Arg Leu               1445                1450                1455 Glu Val LeuGly Cys Glu Ala Gln Asp Leu Tyr            1460                1465

Preferably, the surfactant will be a non-ionic surfactant. Non-ionicsurfactants include notably polysorbates and block copolymers likepoloxamers (i.e. copolymers of polyethylene and propylene glycol).According to a preferred variant of the invention, the surfactant willbe a polysorbate. More preferably, a polysorbate included in acomposition according to the instant invention will have a meanpolymerisation degree of from 20 to 100 monomer units (preferably about80), and may for example be polysorbate 80. Preferably also, thepolysorbate should be vegetable-derived.

Preferably, the buffer devoid of amino acids will betris(hydroxymethyl)methylamine (hereafter abridged “tris”).

Preferably also, the pH of the pharmaceutical composition prior tolyophilisation and after reconstitution in water for injection will befrom 6.5 to 7.5, and more preferably about 7.0.

Preferably, a solid composition according to the invention will be suchthat it may be obtained by lyophilisation of a solution devoid of aminoacids that comprises:

-   (a) a concentration of factor VIII ranging from 50 to 10,000    international units/ml for human or recombinant human factor VIII or    from 50 to 10,000 porcine units/ml for porcine or recombinant    porcine factor VIII;-   (b) a concentration of surfactant ranging from above critical    micellar concentration to 1% v/v;-   (c) a concentration of calcium chloride ranging from 0.5 to 10 mM;-   (d) a concentration of sucrose ranging from 5 to 50 mM;-   (e) a concentration of sodium chloride ranging from 0.15 to 0.5 M;-   (f) a concentration of trisodium citrate ranging from 1 to 50 mM;    and-   (g) a concentration of buffer devoid of amino acids ranging from 1    to 50 mM.

For evaluating the activity in terms of international factor VIII units,the product to be tested is assayed against a Concentrate Standard, suchas the United Kingdom standard NIBSC 95/608 (NIBSC for NationalInstitute of Biological Standards and Control).

By porcine unit of factor VIII is meant the United Kingdom nationalstandard unit held by United Kingdom's NIBSC. For evaluating theactivity in terms of porcine factor VIII units, the product to be testedis assayed against the UK national porcine standard NIBSC 86/514.Concerning recombinant porcine factor VIII, it should be understood that1 unit of activity for recombinant porcine factor VIII is equivalent to1 unit of activity for porcine factor VIII.

More preferably, a solid composition according to the invention will besuch that it may be obtained by lyophilisation of a solution devoid ofamino acids that comprises at least one of the followingcharacteristics:

-   -   a concentration of factor VIII ranging from 100 to 5,000        international units/ml for human or recombinant human factor        VIII or from 100 to 5,000 porcine units/ml for porcine or        recombinant porcine factor VIII;    -   a concentration of surfactant ranging from 0.002% to 0.04% v/v;    -   a concentration of calcium chloride ranging from 1 to 5 mM;    -   a concentration of sucrose ranging from 5 to 25 mM;    -   a concentration of sodium chloride ranging from 0.2 to 0.4 M;    -   a concentration of trisodium citrate ranging from 1 to 20 mM; or    -   a concentration of buffer devoid of amino acids ranging from 1        to 20 mM.

Even more preferably, a solid composition according to the inventionwill be such that it may be obtained by lyophilisation of a solutiondevoid of amino acids that comprises at least one of the followingcharacteristics:

-   -   a concentration of factor VIII ranging from 200 to 2,000        international units/ml (and notably about 1,000 international        units/ml) for human or recombinant human factor VIII or from 200        to 2,000 porcine units/ml (and notably about 1,000 porcine        units/ml) for porcine or recombinant porcine factor VIII;    -   a concentration of surfactant ranging from about 0.002% to 0.02%        v/v (and notably about 0.01% v/v);    -   a concentration of calcium chloride ranging from 1 to 3 mM (and        notably about 2 mM);    -   a concentration of sucrose ranging from 5 to 15 mM (and notably        about 11.7 mM);    -   a concentration of sodium chloride ranging from 0.25 to 0.35 M        (and notably about 0.3 M);    -   a concentration of trisodium citrate ranging from 1 to 20 mM        (and notably about 10 mM); or    -   a concentration of buffer devoid of amino acids ranging from 5        to 15 mM (and notably about 10 mM).

The solid factor VIII compositions according to the invention may beprepared by lyophilising a solution comprising the appropriatequantities of the components identified above as (a), (b), (c), (d),(e), (f) and (g) according to standard manufacturing procedures (sterileconditions, etc.).

Stability of the composition over a certain period may be determined,for example, by the method described hereunder in the part entitled“Analytical methods”, or by any other method found appropriate by theskilled artisan.

A composition according to the invention is considered stable during acertain period of time if 70% to 130% (and preferably 80% to 120%) ofthe initial factor VIII activity, as evaluated using the methoddisclosed the part entitled “Analytical methods” hereafter, ismaintained over said period of time.

Preferably, the solid compositions of this invention will be stable forat least 6 or 12 months when kept at a temperature of 2 to 8° C. Morepreferably, they will be stable for at least 6 or 12 months when kept ata temperature of 30 to 32° C.

The solid factor Vm compositions according to the invention may bediluted with sterile water optionally containing sodium chloride, andthe resulting liquid pharmaceutical composition may then be directlyinjected into a patient in need thereof. The resulting liquidpharmaceutical composition, as well as liquid pharmaceuticalcompositions obtainable by dilution of solid factor VIII compositionsaccording to the invention with sterile water optionally containingsodium chloride, are also part of this invention.

Methods of treatment of haemophilia comprising the administration of aliquid composition according to the invention to a patient in needthereof are also within the scope of this invention. The administrationmode contemplated for liquid compositions according to the instantinvention will preferably be intravenous administration. The dose ofcomposition according to the instant invention which is to beadministered will be determined by the treating physician orveterinarian, taking into account the severity of the disease for eachpatient.

The term “about” refers to an interval around the considered value. Asused in this patent application, “about X” means an interval from Xminus 10% of X to X plus 10% of X, and preferably an interval from Xminus 5% of X to X plus 5% of X.

Unless they are defined differently, all the technical and scientificterms used here have the same meaning as that usually understood by anordinary specialist in the field to which this invention belongs.Similarly, all publications, patent applications, all patents and allother references mentioned here are incorporated by way of reference.

The following examples are presented to illustrate the above and must inno case be considered as a limit to the scope of the invention.

EXAMPLES Example 1

A solution in 0.5 ml sterile water containing the following componentsis prepared: Modified porcine factor VIII of 800 porcine units/mlsequence SEQ. ID. NO. 1 Vegetable derived polysorbate 80 0.01% v/vCalcium chloride 2 mM Sucrose 11.7 mM Sodium chloride 0.3 M Tri sodiumcitrate 10 mM Tris buffer 10 mM pH 7.0

The mixture is lyophilised in a sterilised vial which is then sealed.The solid composition obtained has been tested and shown to be stable ata temperature of 2 to 8° C. for at least 18 months and at 30 to 32° C.for at least six months when tested by factor VIII activity. There wasno indication of high molecular weight component formation as assessedby Size Exclusion HPLC (SEC HPLC) or fragments as assessed by SDS PAGE.

The lyophilised mixture obtained would typically be reconstituted with1.0 ml sterile water before injection into a patient.

Example 2

A solution in 1.0 ml sterile water containing the following componentsis prepared: Modified porcine factor VIII of 400 porcine units/mlsequence SEQ. ID. NO. 1 Vegetable derived polysorbate 80 0.002% v/vCalcium chloride 2 mM Sucrose 11.7 mM Sodium chloride 0.3 M Tri sodiumcitrate 10 mM Tris buffer 10 mM pH 7.0

The mixture is lyophilised in a sterilised vial which is then sealed.

The lyophilised mixture obtained would typically be reconstituted with2.0 ml sterile water before injection into a patient.

Example 3

A solution in 0.5 ml sterile water containing the following componentsis prepared: Plasma-derived porcine factor VIII 100 porcine units/mlVegetable derived polysorbate 80 0.01% v/v Calcium chloride 2 mM Sucrose11.7 mM Sodium chloride 0.3 M Tri sodium citrate 10 mM Tris buffer 10 mMpH 7.0

The mixture is lyophilised in a sterilised vial which is then sealed.

The lyophilised mixture obtained would typically be reconstituted with1.0 ml sterile water before injection into a patient.

Analytical Methods

Chromogenic Assay

The factor VIII activity is determined by a modified chromogenic assay(Technochrom FVIII:C Reagent Kit, Technoclone). The generation ofactivated factor X by factor IX is stimulated by factor VIII which actsas a cofactor in the reaction. The release of p-nitroaniline from thechromogenic substrate is catalysed by activated factor X. The amount ofp-nitroaniine which is released is measured photometrically at 405 nmand the assay gives a linear correlation between the amount ofp-nitroaniline generated and the FVIII content.

SEC HPLC

Soluble high molecular weight components and fragments were determinedby gel filtration performed on a HPLC instrument using a TosoHaas TSKG3000 SVXL, 0.78×30 cm pre-packed column with a fluorescence detector(Waters LC Module 1 plus). Excitation wavelength 280 nm and emissionwavelength 340 nm. Evaluation of the results were performed byintegration of the peak areas.

SDS PAGE Assay

SDS PAGE (polyacylamide gel electrophoresis using a flatbedelectrophoresis system (Multiphor II LKB) and pre cast 7.5% gels(EXCELGEL SDS, Pharmacia) was used to determine any breakdown productsof the FVIII molecule. Protein bands were visualised by Coomassie bluestaining.

Stability Assay

Stability can be assayed by performing the above described assays atdifferent times on a sample of the same composition held at thetemperature chosen (which may be around +4° C. or +31° C.). Once itsfactor VIII activity will have dropped of more than 30%, the compositionwill be considered to have lost its stability.

1. A solid pharmaceutical composition obtainable by lyophilization of asolution devoid of amino acids comprising (a) factor VIII; (b) asurfactant; (c) calcium chloride; (d) sucrose; (e) sodium chloride; (f)trisodium citrate; and (g) a buffer devoid of amino acids; having a pHfrom 6 to 8 prior to lyophilization and after reconstitution in waterfor injection.
 2. A solid pharmaceutical composition to of claim 1wherein the factor VIII is porcine factor VIII or recombinant porcinefactor VIII.
 3. A solid pharmaceutical composition of claim 2 whereinthe factor VIII is recombinant porcine factor VIII.
 4. A solidpharmaceutical composition of claim 2 wherein the recombinant porcinefactor VIII has the amino acid sequence SEQ. ID. NO.
 1. 5. A solidpharmaceutical composition of claim 1 wherein the surfactant is apolysorbate.
 6. A solid pharmaceutical composition of claim 5 whereinthe surfactant is a polysorbate
 80. 7. A solid pharmaceuticalcomposition of claim 1 wherein the buffer devoid of amino acids istris(hydroxymethyl)methylamine.
 8. A solid pharmaceutical composition ofclaim 1 wherein prior to lyophilization and after reconstitution inwater for injection, has a pH from 6.5 to 7.5.
 9. A solid pharmaceuticalcomposition of claim 1 which may be obtained by of a solution devoid ofamino acids that comprises: (a) a concentration of factor VIII from 50to 10,000 international units/ml for human or recombinant human factorVIII or from 50 to 10,000 porcine units/ml for porcine or recombinantporcine factor VIII; (b) a concentration of surfactant from abovecritical micellar concentration to 1% v/v; (c) a concentration ofcalcium chloride from 0.5 to 10 mM; (d) a concentration of sucrose from5 to 50 mM; (e) a concentration of sodium chloride from 0.15 to 0.5 M;(f) a concentration of trisodium citrate from 1 to 50 mM; and (g) aconcentration of a buffer devoid of amino acids from 1 to 50 mM.
 10. Aliquid pharmaceutical composition obtainable after dilution of a solidpharmaceutical composition of claim 1 with sterile water optionallycontaining sodium chloride.